Use of cellular extracts for obtaining pluripotent stem cells

ABSTRACT

The use of a composition including at least one permeabilized nucleus of a first cell, or at least one permeabilized first cell including the nucleus and an extract of female germinal cells, or eggs, of a multicellular organism, the eggs being blocked in the metaphase II of meiosis, the extract including EGTA, for carrying out a method for obtaining pluripotent stem cells, or tissues derived from the pluripotent stem cells, or of cloning, provided that the process is not for cloning human beings.

The present invention relates to the use of cellular extracts forobtaining pluripotent stem cells, in particular for reprogrammingdifferentiated cells.

Nuclear transfer is a powerful method that can be used to obtain newsources of multipotential cells from differentiated tissues and toproduce cloned animals. By transplanting nuclei from differentiatedamphibian or mammalian cells into enucleated eggs, blastula orblastocyst embryos can be obtained which can develop into entire animalsor used to form a wide range of tissues and cell types [Gurdon, et al.(2003). Proc Natl Acad Sci USA 100 Suppl 1, 11819-11822]. The potentialability to deliver supplies of multipotential cells, which hold greatpromise for cell-based therapies for numerous disorders, makes nucleartransfer an appealing alternative to the difficult practice of directlyisolating natural stem cells from normal adult tissues [McKay, R.(2000). Stem cells—hype and hope. Nature 406, 361-364].

It has been recently suggested that different reprogramming strategiescould be associated together to synergize their efficiencies [Gurdon J &Murdoch A (2008) Cell Stem Cell 2:135-138]. Several attempts have beenmade by using cellular extracts to reprogram somatic cells, but theyfailed to reproduce the range of effects obtained by NT.

In NT experiments, it is the exposure of transplanted nuclei to thecytoplasm of the receiving oocyte that induces the reprogramming. Sinceeggs naturally contain all the genetic and epigenetic factors essentialfor totipotency, embryonic stem cells obtained by nuclear transfer arecloser to natural embryonic stem cells. However, this is hard to mimicin vitro due to the difficulty to obtain large quantities of mammalianoocytes.

Despite its many advantages, however, nuclear transplantation is ofteninefficient due to the difficulty involved in completely reprogrammingdifferentiated adult nuclei for the events of early development. Indeed,it is known that the ability of the egg to reset the epigenetic marks ofadult donor cells is determinant for the efficiency of nuclear cloning.Identifying the specific epigenetic properties of differentiated cellnuclei that must be reset before development can begin anew, and howsuch resetting can be efficiently achieved, thus represents a challengeof major biological and medical significance.

First Generation

Various methods have been identified that can enhance the efficiency ofnuclear transplantation.

In amphibians, for instance, cloning efficiency is substantiallyimproved by serial nuclear transfers. This consists of transferring anucleus from a differentiated donor cell to an enucleated egg, allowingthe cell to undergo several divisions, and then using the daughternuclei as donors for a second nuclear transfer experiment [Gurdon, J. B.(1962). J Embryol Exp Morphol 10, 622-640]. Injections of nuclei intomaturing oocytes instead of eggs [DiBerardino, M. A., and Hoffner, N. J.(1983) Science 219, 862-864] led to the hypothesis that components ofmaturing oocytes may enable the injected nucleus to respond to DNAsynthesis-inducing factors in activated eggs [Leonard, et al. (1982) DevBiol 92, 343-355]. One possible factor contributing to the lowefficiency of cloning experiments is that the chromosome organization ofdifferentiated adult nuclei may not be well adapted for DNA replication.

Animal cloning represents a major challenge in various fields, from theconservation of animal species, the production of proteins, such astherapeutic proteins, by cloned animals, to the therapeutic cloning,particularly for obtaining stems cells useful for autologoustransplants.

However, the efficiency of the current cloning techniques needs to beimproved to in order to contemplate large scale applications.

The deficiency of the prior art have been partially solved.

The international application WO 2007/039258 discloses the use of acellular extract for remodeling chromosomes, in order to carry out aprocess for reprogramming, or cloning cells. The aim of this applicationis essentially to reprogram chromosomes. Thus, this document onlypartially solves the problem regarding the complete dedifferentiation ofdifferentiated cells.

So the techniques of NT needed to be improved.

Second Generation

More recently, other approaches have been made in order to enhance theefficiency of cloning and the efficiency of the method of reprogrammingdifferentiated cells.

For instance, the European patent application EP 1 970 446 discloses theinduction of pluripotency, in somatic cells, by the ectopic expressionof the four transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM).This document demonstrate that the above four factors allow to obtaininduced pluripotent stem (iPS) cells, which are highly similar toembryonic stem (ES) cells.

Notably, murine iPS cells have a complete developmental potential asdemonstrated by their capacity to form teratomas, generate chimeras andcontribute to the germline. However, the efficiencies of both iPS cellproduction and nuclear transfer (NT) remain low and most of the obtainedreprogrammed cells appear to be only partially reprogrammed.

Thus, additional factors may be needed to improve them [Feng B, et al.(2009) Cell Stem Cell 4:301-312; Huangfu D, et al. (2008) Nat Biotechnol26:795-797] and many efforts have been done over the last years tooptimize these procedures.

A recent review [Lai et al. (2011) J. Assist Reprod Genet, Mar. 9, 2011]summarizes the improvements of the iPS technologies since its firstdisclosure (see EP1970446), and conclude that up to date, manychallenges need to be overcome, in particular regarding the efficiency.

Therefore, there is a need to provide improvement of the iPStechnologies to avoid the deficiencies of the methods disclosed in theart.

The aim of the invention is to overcome such deficiencies.

One particular aim of the invention is to provide a new method thatincreases the efficiency of dedifferentiation of differentiated cells.

Another aim of the present invention is to provide a compositioncarrying out said method. Another aim of the invention is also toprovide a process for obtaining multipotent or totipotent stem cells ofpluricellular organisms.

The invention relates to the use of a composition comprising:

-   -   at least one permeabilized nucleus of a first cell, or at least        one permeabilized first cell comprising said nucleus

said first cell being an induced pluripotent stem cell, i.e. iPS cell,originating from a multicellular organism,

and

-   -   an isolated extract of female germinal cells, or of eggs, of a        multicellular organism, said eggs being blocked in the metaphase        II of meiosis, said extract comprising EGTA,

for carrying out a method

-   -   for obtaining pluripotent stem cells, or tissues derived from        said pluripotent stem cells, or    -   of cloning, in particular, of cloning provided that said process        is not for cloning human beings.

The invention is based on the unexpected observation made by theInventors that the combination of cellular extracts of eggs ofpluricellular cells and genes allowing the dedifferentiation ofdifferentiated cells act synergistically to significantly enhance thecell reprogramming, i.e. cellular dedifferentiation of differentiatedcells, and thus enhance nuclear transfer and cloning obtained by thetechniques of the prior art: iPS and nuclear extract.

The expression “germinal cell” refers to a cell susceptible to form thegametes.

The expression “female germinal cell”, also called “egg” relates a cellat any stage of the oogenesis, particularly primordial germ cells,oogonia and oocytes.

The germinal cell extract is made from eggs which are arrested at themetaphase stage of the second meiotic division.

The “extract of female germinal cells, or eggs” is a cell extractobtained by the implementation of the process as described in Menut etal. [Menut et at (1999) Advances in Molecular Biology: A comparativeMethods Approach to the Study of Ooocytes and Embryos, ed Richter J D(OxfordUniversity Press), pp 196-226, 2001] (referred as CSF extract).

The above extract is “isolated”, which means that said extract isobtained in vitro. This extract is thus completely different from a cellor a part of cell, such as spheroplast or liposomes, which are known inthe art.

Extracts are prepared as disclosed in Example 1.

One of the absolute requisite in the extract is the presence of EGTA, acalcium chelating agent which allows the maintaining of the Extract inthe metaphase II of the meiosis. Indeed, it is well known in the artthat eggs are activated, and exits from metaphase II when calcium entersin cell, following the contact with spermatozoid.

By extension, it is also known that electric chock, needle contact, orany action modifying the plasma membrane of the egg will induce calciumflux in egg, and thus will “unlock” the egg which became activatedmetaphase II arrested egg: i.e. an interphase egg.

In the invention, it is fundamental that the isolated extract of femalegerminal cells, or the isolated extract of eggs, is kept in a metaphasicstate during its preparation and its use.

It is necessary to control the fragile state induced by EGTA, in whichthe isolated extract of female germinal cells, or the isolated extractof eggs, is blocked in metaphase. In the invention, the blockage of theisolated extract in metaphase is maintained stable, i.e. the eggs usedto obtain the extract of female germinal cells, or of eggs, of amulticellular organism are blocked, in a stable way, in the metaphase IIof meiosis.

The stability of the blockage of the isolated extract in metaphase isdetermined by different ways. Metaphase stage of the extracts can bechecked by the structure of the chromatin, the phosphorylation ofhistone H3 on serine 10. Moreover, the metaphasic stability of theextracts can be followed up of DNA synthesis since extracts blocked inmetaphase can replicate efficiently sperm chromatin only if they arepreviously activated by calcium addition. Tests of the follow up DNAsynthesis are achieved by techniques well known in the art, such asmeasuring [³²P]αdCTP incorporation (FIG. 12).

In what precedes and what follows, the female germinal cell extract canbe replaced by a mitotic non-human early embryo of vertebrates. Saidmitotic non-human early embryo of vertebrates may be obtained by theprocess described in Lemaitre et al. [Lemaitre et at (2005) Cell123:1-15].

The expression “pluricellular organism” (or “multicellular organism”)refers to living organisms that are composed of several cells. In saidmulticellular or pluricellular organisms, the similar cells usuallyaggregate in tissues and the specific arrangements of different tissuesform organs.

In the invention “induced pluripotent cells” commonly abbreviated as iPScells or iPSCs refers a type of pluripotent stem cell artificiallyderived from a non-pluripotent cell, typically an adult somatic cell, byinducing a “forced” expression of specific genes.

Induced pluripotent stem cells (iPS cells) are similar to naturalpluripotent stem cells, such as embryonic stem (ES) cells, in manyrespects, such as the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability, but the full extent of their relation to naturalpluripotent stem cells is still being assessed.

One of the unexpected observations made by the Inventors is thatheterogous extract can synergize with iPS inducing genes todedifferentiate differentiated cells. In other words, Xenopus extract,with iPS inducing genes, can act to dedifferentiate differentiated cellsoriginating from multicellular organisms different from Xenopus.

Mutatis mutandis, mouse egg extracts could synergize with iPS inducinggenes to dedifferentiate, for instance Xenopus differentiated cells.

All the combinations are possible, and the skilled person, without undueburden could easily choose the extract which is easier for him to carryout the method according to the invention.

Stem cells are primal undifferentiated cells that retain the ability todivide and can differentiate into other cell types. Totipotent stemcells can differentiate into embryonic and extra-embryonic cell types.Pluripotent stem cells originate from totipotent cells and can give riseto progeny that are derivatives of the three embryonic germ layers,mesoderm, ectoderm and endoderm.

Somatic cells are any cells other than oocytes and spermatozoids.

“Somatic differentiated cells” are somatic cells that are specialized ina particular function and that do not maintain the ability to generateother kinds of cells or to revert back to a less differentiated state.

The differentiated somatic cells may particularly originate from anykind of tissue of the organism, such as skin, intestine, liver, blood,muscle, etc.

To improve the entrance of the female germinal cell extract into thecells, the cell membrane is be permeabilized. The permeabilization ofthe cell membrane is achieved by the techniques well known in the art,such as the use of a chemical agent or a mild detergent, such asdigitonine, Nonidet™ P40 (4-Nonylphenyl-polyethylene glycol; NP40),Triton® X100 (4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tent-octylphenylether), sodium deoxycholate (DOC), Triton® N101 (Polyoxyethylenebranched nonylcyclohexyl ether), Brij® 96 (polyoxyethylene 10 oleoylether), or an enzyme that can make small holes in the cell membrane, aslysolecithin, or via a mechanical process which can at least partly openthe cell membrane, for example pipetting.

Preferably, nucleus of the first cell, or the first cell comprising saidnucleus are lightly permeabilized.

“Lightly permeabilization” is obtained for example by using lowconcentrations of detergent like NP40, Triton® X100, DOC, Triton® N101,Brij® 96, Lysolecithin, digitonin.

In one advantageous embodiment, the invention relates to the use asdefined above, wherein said iPS cell is obtained by allowing theexpression, in a somatic differentiated cell, of with at least an Octfamily member protein and a Sox family member protein, along with atleast one other factor chosen among a Klf family member protein, a Mycfamily member protein, the Nanog member protein and the LIN28 member.

In one advantageous embodiment, the invention relates to the use asdefined above wherein said iPS cell is obtained by transfecting asomatic differentiated cell with genes coding for at least an Oct familymember protein and a Sox family member protein, along with at least oneother factor chosen among a Klf family member protein, a Myc familymember protein, the Nanog member protein and the LIN28 member.

In one advantageous embodiment, the invention relates to the use asdefined above, wherein said iPS cell is obtained by allowing theexpression, in a somatic differentiated cell, of with at least an Octfamily member protein and a Sox family member protein, along with

-   -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog member protein and the LIN28 member.

In one advantageous embodiment, the invention relates to the use asdefined above wherein said iPS cell is obtained by transfecting asomatic differentiated cell with genes coding for at least an Oct familymember protein and a Sox family member protein, along with:

-   -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog member protein and the LIN28 member.

In one another advantageous embodiment, the invention relates to the useas defined above, wherein said Oct family member protein is either theOct3 or the Oct4 protein.

In one another advantageous embodiment, the invention relates to the useas defined above, wherein said Sox family member protein is the Sox2protein.

In one other advantageous embodiment, the invention relates to the useas defined above, wherein said iPS cell is obtained by allowing theexpression, in a somatic differentiated cell, of at least Oct4 proteinand a Sox2 protein, along with at least one other factor chosen among aKlf family member protein, a Myc family member protein, a Nanog memberprotein and a LIN28 family member protein.

In one advantageous embodiment, the invention relates to the use asdefined above wherein said iPS cell is obtained by transfecting asomatic differentiated cell with genes coding for at least an Oct4protein and a Sox 2 protein, along with at least one other factor chosenamong a Klf family member protein, a Myc family member protein, a Nanogmember protein and a LIN28 family member protein.

The Inventors have demonstrated that the following minimal combinationof genes:

-   -   Oct4, Sox2 and a Klf family member protein,    -   Oct4, Sox2 and a Myc family member protein    -   Oct4, Sox2 and a Nanog member protein, and    -   Oct4, Sox2 and a LIN28 family member protein,

in combination with the extract mentioned above, are able to enhance thenuclear transfer and cloning with respect to the genes only or theextract only.

In one another advantageous embodiment, the invention relates to the useas defined above, wherein said Klf family member protein is the Klf4protein.

In one another advantageous embodiment, the invention relates to the useas defined above, wherein said Myc family member protein is the c-Mycprotein.

In still another advantageous embodiment, the invention relates to theuse as defined above, wherein said iPS cell is obtained by the contactof a somatic differentiated cell, with:

-   -   either a first composition Oct4, Sox2, Klf4 and c-Myc proteins,    -   or a second composition Oct4, Sox2, Nanog, and LIN28 proteins.

The above combinations of genes are the preferred ones.

The invention relates, in another advantageous embodiment, to the use ofas defined above, wherein said extract is substantially devoid of anycytoplasmic membrane.

On the contrary to other cloning techniques, consisting of fragmentation(sonication, mechanical fragmentation . . . ) of egg from which thenucleus has been extracted, the extract according to the invention isdevoid of any cytoplasmic membrane. However, the extract may containnuclear membrane precursors (vesicles containing nuclear envelopmembranes) that could be used in order to reconstitute a functionalnucleus.

In still another advantageous embodiment, the invention relates to theuse as defined above, wherein said extract comprises EGTA in aconcentration from 0.1 mM to 10 mM, preferably from 0.5 mM to 6 mM, morepreferably from 1 mM to 4 mM, in particular from 1 to 2 mM.

In one embodiment, the invention relates to the use as defined above,wherein said isolated extract of female germinal cells, or isolatedextract of eggs, are non-human cells.

The invention also relates to a composition comprising:

-   -   at least one permeabilized nucleus of a first cell, or at least        one permeabilized first cell comprising said nucleus

said first cell originating from a multicellular organism, said firstcell being an induced pluripotent stem cell, i.e. iPS cell, and

-   -   a, possibly isolated, extract of female germinal cells, or of        eggs, of a multicellular organism, said eggs being blocked in        the metaphase II of meiosis, said extract comprising EGTA.

The nuclei can be extracted from the cells by the techniques well knownin the art, such as cell breakage by incubation in a hypotonic buffer,use of a dounce homogeneizer or a potter homogeneizer or an isotonicbuffer containing sucrose, glycerol or similar stabilizing agent and useof a potter homogeneizer or dounce homogeneizer able to open or disruptthe cell membrane.

The nuclei are either used directly or stored in specific conditions tomaintain their integrity, such as storage at −20° C., −80° C. or inliquid nitrogen in conditions known to be used to store oocytes or earlyembryos.

In one advantageous embodiment, the invention relates to the compositionas defined above, wherein said iPS cell is obtained by the contact of asomatic differentiated cell, with at least:

-   -   an Oct family member protein and a Sox family member protein,        and    -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog member protein and the LIN28 member.    -   In one advantageous embodiment, the invention relates to a        composition as defined above wherein said composition comprises:    -   at least one permeabilized nucleus of a first cell, or at least        one permeabilized first cell comprising said nucleus

said first cell originating from a multicellular organism, said firstcell, being an induced pluripotent stem cell, i.e. iPS cell, said iPScell expressing a combination of genes coding for at least:

-   -   an Oct family member protein and a Sox family member protein,        and    -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog member protein and the LIN28 member.    -   and    -   an isolated extract of female germinal cells, or of eggs, of a        multicellular organism, said eggs being blocked in the metaphase        II of meiosis, said extract comprising EGTA.

In one another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said Oct family member protein iseither the Oct3 or the Oct4 protein.

In one another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said Sox family member protein isthe Sox2 protein.

In one another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said Klf family member protein isthe Klf4 protein.

In one another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said Myc family member protein isthe c-Myc protein.

In still another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said iPS cell is obtained by thecontact of a somatic differentiated cell, with the four proteins Oct4,Sox2, Klf4 and c-Myc proteins, or with the four proteins Oct4, Sox2,Nanog, and LIN28 proteins.

In one another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said iPS cell is obtained bytransfecting a somatic differentiated cell with genes coding the fourproteins Oct4, Sox2, Klf4 and c-Myc proteins, or the four proteins Oct4,Sox2, Nanog, and LIN28 proteins.

Both of said four proteins have similar effects regarding theirsynergistic effect when used with the extract as defined in theinvention.

The invention relates, in another advantageous embodiment, to thecomposition of as defined above, wherein said extract said extract issubstantially devoid of any lipid membrane.

In still another advantageous embodiment, the invention relates to thecomposition as defined above, wherein said extract comprise EGTA in aconcentration from 0.1 mM to 10 mM, preferably from 0.5 mM to 6 mM, morepreferably from 1 mM to 4 mM, in particular from 1 to 2 mM.

In one advantageous embodiment, the invention relates to a compositionas defined above, wherein said female germinal cells are vertebratefemale germinal cells, preferably chosen among mammals, in particularhumans, birds, reptiles and amphibians.

The techniques for obtaining egg, or female germinal cells, from theabove vertebrate are current veterinary techniques well known in theart.

In one another advantageous embodiment, the invention relates to acomposition as defined above, wherein said female germinal cells areXenopus cells.

The Xenopus eggs are obtained as disclosed in Menut et al. 1999.

In one embodiment, the invention relates to a composition as definedabove, wherein said isolated extract of female germinal cells, orisolated extract of eggs, are non-human cells.

The invention also relates to a method for producing pluripotent stemcells, comprising a step of contacting at least one permeabilizednucleus of a first cell, or at least one permeabilized first cellcomprising said nucleus, said first cell originating from amulticellular organism, said first cell, being an induced pluripotentstem cell, i.e. iPS cell, with an (isolated) extract of female germinalcells, or of eggs, of multicellular organism, said eggs being blocked inthe metaphase II of meiosis, said extract comprising EGTA.

The invention also relates to a method for producing pluripotent stemcells, comprising a step of contacting at least one permeabilizednucleus of a first cell, or at least one permeabilized first cellcomprising said nucleus,

said first cell originating from a multicellular organism, said firstcell being an induced pluripotent stem cell, i.e. iPS cell,

with an extract of female germinal cells, or of eggs, of multicellularorganism, said eggs being blocked in the metaphase II of meiosis, saidextract comprising EGTA,

possibly provided that said method is not a process for cloning humanbeings.

The term “contacting” means that the cells, or nuclei, and the femalegerminal extract are present together in suitable conditions, in orderto allow the diffusion of the molecules contained in the female germinalextract into said cells or nuclei. The contact is carried out preferablyat a temperature preferably comprised from 20° C. to 23° C., andpreferentially for at least 10 minutes, more preferably at least 20minutes, more preferably at least 30 min.

According to the method mentioned above, permeabilized nuclei of adifferentiated cells, or permeabilized differentiated cells, after acontact with the extract of female germinal cells, or of eggs, ofmulticellular organism, said eggs being blocked in the metaphase II ofmeiosis, said extract comprising EGTA, have acquire, or re-acquire allthe features of a pluripotent stem cell.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said iPS cell is obtained by the contact of asomatic differentiated cell, with at least:

-   -   an Oct family member protein and a Sox family member protein,        and    -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog family member protein and the LIN28 family member        protein.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said iPS cell is obtained by transfecting asomatic differentiated cell, with genes coding at least:

-   -   an Oct family member protein and a Sox family member protein,        and    -   either a Klf family member protein and a Myc family member        protein,    -   or the Nanog family member protein and the LIN28 family member        protein.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said Oct family member protein is either the Oct3or the Oct4 protein.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said Sox family member protein is the Sox2protein.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said Klf family member protein is the Klf4protein.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said Myc family member protein is the c-Mycprotein.

Thus, in the case of the permeabilized cells, if the cells aremaintained in an appropriate culture medium, preferably a culture mediumused for maintaining ES cells, said cells will harbour similar featureswith respect to the ES cells, after the contact with the extract, andthe re-expression of the above genes, i.e. Oct4, Sox2, Klf4 and c-Mycproteins or Oct4, Sox2, Nanog, and LIN28 proteins.

In one advantageous embodiment, the invention relates the method asdefined above wherein, in the first step of transfecting adifferentiated somatic cell with genes coding for at least:

-   -   either a first combination of Oct4, Sox2, Klf4 and c-Myc        proteins,    -   or a second combination of Oct4, Sox2, Nanog, and LIN28        proteins,    -   the first combination or the second combination comprises        respectively at least nucleic acid coding for Oct4, Sox2, Klf4        and c-Myc proteins, or Oct4, Sox2, Nanog, and LIN28 proteins.

In one advantageous embodiment, the invention relates to the methoddefined above, said method comprising

-   -   a step of contacting a differentiated somatic cell with        -   either a first composition Oct4, Sox2, Klf4 and c-Myc            proteins,        -   or a second composition Oct4, Sox2, Nanog, and LIN28            proteins,    -   to obtain a first cell which is an iPS cell,    -   a step of permeabilizing said first cell obtained in the        previous step, to obtain permebilised iPS cells and    -   a step of contacting said permeabilized iPS cell with an        (isolated) extract of female germinal cells, or eggs, of        multicellular organism, said eggs being blocked in the metaphase        II of meiosis, said extract comprising EGTA.

In one advantageous embodiment, the invention relates to the methoddefined above, said method comprising

-   -   a step of transfecting a differentiated somatic cell with genes        coding for        -   either a first combination of Oct4, Sox2, Klf4 and c-Myc            proteins,        -   or a second combination Oct4, Sox2, Nanog, and LIN28            proteins,    -   to obtain a first cell which is an iPS cell,    -   a step of permeabilizing said first cell obtained in the        previous step, to obtain permeabilised iPS cells and    -   a step of contating said permeabilized iPS cell with an isolated        extract of female germinal cells, or of eggs, of multicellular        organism, said eggs being blocked in the metaphase II of        meiosis, said extract comprising EGTA.

In one advantageous embodiment, the invention relates to the methoddefined above, wherein said female germinal cells are vertebrate femalegerminal cells, preferably chosen among mammals, in particular humans,birds, reptiles and amphibians, preferably said germinal cells areXenopus cells.

In one embodiment, the invention relates to the method as defined above,wherein said isolated extract of female germinal cells, or said isolatedextract of eggs, are non-human cells.

The invention also relates to the pluripotent stem cells liable to beobtained by the process according to the method previously defined.

The Inventors have demonstrated that the cells obtained by the processaccording to the invention harbour an epigenetic pattern that differsfrom the pattern of the natural stem cells.

In one advantageous embodiment, the invention relates to the methoddefined above, wherein, in the step of contacting a differentiatedsomatic cell with

-   -   either a first composition Oct4, Sox2, Klf4 and c-Myc proteins,    -   or a second composition Oct4, Sox2, Nanog, and LIN28 proteins,

the first composition or the second composition comprise respectively atleast a nucleic acid coding for Oct4, Sox2, Klf4 and c-Myc proteins, orOct4, Sox2, Nanog, and LIN28 proteins.

In one advantageous embodiment, the invention relates to the methoddefined above, wherein said at least nucleic acid coding for Oct4, Sox2,Klf4 and c-Myc proteins, or Oct4, Sox2, Nanog, and LIN28 proteins iscomprised in at least one viral vector, preferably in at least oneretroviral vector, in particular in at least one integrative retroviralvector or a lentiviral vector.

In one advantageous embodiment, the invention relates to the methoddefined above, wherein said at least one integrative retroviral vectoris integrated into the genome of said differentiated somatic cell.

In one advantageous embodiment, the invention relates to the methoddefined above, said method further comprising a step of culturing thecells obtained in the previous step in a medium allowing maintaining thepluripotency of said pluripotent stem cells.

“Medium allowing maintaining the pluripotency of said pluripotent stemcells” is a medium containing nutriments, growth factors, hormones . . .that allows the cellular division of the undifferentiated cells.

For instance, ES cells are grown at 37° C./5% CO2/95% humidity in dishescoated with a feeder layer of mitotically inactivated primary mouseembryonic fibroblasts, in a DMEM (high glucose, Gibco 41966-052, storein fridge) minimal medium supplemented before use with:

-   -   15% (v/v) FCS (serum from newborn calf, liquid, tested for ES        culture, store aliquots at −20° C.)    -   1/100 (v/v) L-glutamine (200 mM: Gibco 25030-024, store aliquots        at −20° C.), stable in solution for 10 d only    -   1/100 (v/v) non-essential amino acids (Gibco 11140-035, store        aliquots in fridge)    -   1/100 (v/v) pen/strep (Gibco 15140-122, store aliquots at −20°        C.)    -   1/500 (v/v) 2-mercaptoethanol (Gibco 31350-010, 0.1 mM final        conc., store aliquots in fridge)    -   1/10,000 (v/v) LIF (leukemia Inhibiting Factor) (“ESGRO” from        Chemicon, No. ESG1107, 1000 U/ml final conc. or less (depending        on properties of respective cell line), make up 1/100 dilution        in DMEM with 10% (v/v) or so serum to be further diluted by        1/100 to achieve the working conc., store at −20° C.).

The skilled person can easily adapt the above protocol.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said extract is substantially devoid of anyplasma membrane.

In one advantageous embodiment, the invention relates to the method asdefined above, wherein said extract comprise EGTA in a concentrationfrom 0.1 mM to 10 mM, preferably from 0.5 mM to 6 mM, more preferablyfrom 1 mM to 4 mM, in particular from 1 to 2 mM.

The invention also relates to a method for cloning animals, preferablymammals, comprising

-   -   a step of producing pluripotent stem cell as defined in the        method according to the previous definitions    -   a step of transferring the nucleus of said pluripotent stem cell        into an enucleated egg of an animal of the same species as the        species from which said pluripotent stem cell derives, to obtain        an allo-nucleated egg, and    -   a step of transferring said allo-nucleated egg in a        pseudopregnant female of the same species as the species from        which said pluripotent stem cell derives.

In this advantageous embodiment, the method for cloning animal accordingto the invention is as follows:

-   -   differentiated cells are dedifferentiated by expressing the        genes as mentioned above.    -   nuclei of said cells are extracted, slightly permeabilized, and        exposed to the isolated extract of female germinal cells, or of        eggs, of multicellular organism, said eggs being blocked in the        metaphase II of meiosis, said extract comprising EGTA,    -   the nuclei are then optionally washed with PBS,    -   the nuclei are transferred in an enucleated eggs.

The enucleated eggs are obtained by removing the nucleus of the egg bytechniques well known in the art. Thus, the nuclei treated by theextracts according to the invention are micro injected in saidenucleated eggs. Preferably, the nuclei treated according to theinvention are micro injected in enucleated eggs of the same species. Forinstance a mouse nucleus is injected in a mouse enucleated egg. Thenewly obtained nucleated egg is then called allo-nucleated egg.

However, it is possible to micro inject the nuclei into an enucleatedegg of a different species witch is not completely different. Forinstance a mouse nucleus is injected in a rat enucleated egg, and viceversa. The newly obtained nucleated egg is then called hetero-nucleatedegg.

-   -   the newly obtained nucleated egg (hetero or allo nucleated egg)        is thus transferred into the uterus of a pseudopregnant female        of the same species as the species from which the enucleated egg        derives.

Therefore, if the enucleated egg is obtained from a mouse, the pseudopregnant female will be a mice. If the enucleated egg is obtained from arat, the pseudo pregnant female will be a rat.

Progeny obtained from said pseudo pregnant female will be then clones ofthe animal which have given the nuclei treated according to theinvention.

The invention also relates to a method for cloning animals, preferablymammals, comprising

-   -   a step of producing pluripotent stem cells as defined in the        method as defined above,    -   a step of transferring the nucleus of said pluripotent stem        cells in an enucleated egg of an animal of the same species as        the species from which said pluripotent stem cells derive, to        obtain an allo-nucleated egg,

preferably, provided that said method is not a process for cloning humanbeings.

The invention also relates to animals liable to be obtained by theprocess according to the method previously defined.

In the context of the invention, the term “cloning” means obtaining anentire animal, or embryo of said animal, from the nuclei of a donorcell.

The nuclei of the donor cells can be activated to trigger the S-phase ofthe cell cycle, in order to initiate the first divisions of the earlyembryonic development. The activation may be achieved by the techniqueswell known in the art.

The egg extract can be partially or totally removed, particularly bywashing the nuclei or the cells containing said nuclei, for example byseveral washings in Phosphate Buffer Saline (PBS).

The nuclei are introduced into an enucleated egg, according to thetechniques well known in the art, such as microinjection.

For each enucleated egg, one nucleus or one donor cell is introduced.

The enucleated egg preferentially originates from the same species asthe nuclei. The enucleated egg is obtained by techniques well known inthe art.

The enucleated egg containing the nuclei can then be transferred into afemale breeder, so as to perform its early-embryonic, embryonic andfetal development.

The cells originate from different tissues of an organism canparticularly be chosen among cells originating from any kind of tissue,such as skin, intestine, liver, blood, muscle, etc.

The invention also relates to a method for obtaining cells, cellularlines or tissues of pluricellular organisms, particularly vertebrates,at the desired stage of differentiation, comprising:

-   -   a step of producing pluripotent stem cells as defined in the        method as defined above,    -   a step of culturing the pluripotent stem cells in appropriate        conditions to obtain cells, cellular lines or tissues, at the        desired stage of differentiation.

The resulting multipotent or totipotent stem cells can be cultured underappropriate conditions to maintain said cells in an undifferentiatedstate.

The skilled person knows from his general knowledge what are thepreferred growth factors, hormones, medium, and stimulations . . . toachieve a specific differentiation toward a specific type of cells, toobtain cells or organs at a different stage, preferably in vitro.

The invention also relates to cellular lines or tissues of pluricellularorganisms, particularly vertebrates, at the desired stage ofdifferentiation, liable to be obtained by the process according to themethod previously defined.

FIGURES

FIGS. 1A-D show that M phase Xenopus egg extracts improve the efficiencyof nuclear transfer and iPS cells production from mammalian fibroblasts.

FIG. 1A is a schematic representation of nuclear transfer experimentsusing MEFs exposed to M phase Xenopus egg extracts (M phase).

FIG. 1B is a graph re presenting the percentage of 2 cells (A), 4-8cells (B) morulas (C) and (blastocystes) resulting from nuclear transferof MEFs (open square), MEFs exposed to M phase (M-Extract; Black square)and MEFs exposed to interphase (I-Extract; black circles) Xenopus eggextracts and normalized to the number of 2 cell-embryos. As control, EScells are represented (black diamond).

FIG. 1C is a schematic representation of iPS cell generation fromOCT4-GFP positive MEFs by ectopic expression of Oct4/Klf4/Sox2/c-Myc(OKSM) followed, or not (mock), by exposure to M phase Xenopus eggextracts.

FIG. 1D represents the number of OCT4-GFP positive colonies relative tonon-permeabilized cells. The effect of exposure to M phase egg extractson the efficiency of iPS cell production was assessed by measuring theproduction of OCT4-GFP positive colonies after exposure to M phase eggextracts (M phase alone, A), OKSM over-expression (OKSM alone, B) andOKSM over-expression followed by exposure to buffer alone (OKSM+mock; C)or to M phase egg extracts (OKSM+M phase; D), in three fully independentexperiments. Error bars represent s.e.m. (n=3).

FIGS. 2A-U show the characterization of the pluripotency of iPS cellsobtained by OKSM overexpression followed by exposure to M phase Xenopusegg extracts

FIGS. 2A-F represent the alkaline phosphatase expression in mock-treatedMEFs (FIG. 2A and FIG. 2D), ES cells (FIG. 2B and FIG. 2E) and iPS cellsinduced by OKSM over-expression and exposure to M phase egg extracts (Mphase iPS; FIG. 2C and FIG. 2F). FIGS. 2D-F represent respectivelyhigher magnification of FIGS. 2A-C.

FIGS. 2G-J represents the morphology (FIG. 2G, FIG. 2I) and GFPexpression (FIG. 2H, FIG. 2J) in M phase iPS cells generated fromOCT4-GFP MEFs. FIGS. 2I-J-F represent respectively higher magnificationof FIGS. 2G-H.

FIGS. 2K-S represent the expression of pluripotency markers assessed byimmunofluorescence in M phase iPS cells: OCT4 (FIG. 2K), Nanog (FIG. 2N)and SSEA1 (FIG. 2Q) co-localized with GFP whose expression was driven bythe promoter of OCT4 (FIG. 2L, FIG. 2O and FIG. 2R). DNA is labelledwith DAPI (FIG. 2M, FIG. 2P and FIG. 2S).

FIG. 2T represents the expression of Oct4 measured by quantitativeRT-PCR in MEFs (first column), ES cells (second column) and two M phaseiPS clones (M/iPS; two last columns). Error bars represent s.e.m. (n=3).Y-axis represents the expression of mRNA relative to ES cells.

FIG. 2U represents the expression of Nanog measured by quantitativeRT-PCR in MEFs (first column), ES cells (second column) and two M phaseiPS clones (M/iPS; two last columns). Error bars represent s.e.m. (n=3).Y-axis represents the expression of mRNA relative to ES cells.

FIGS. 3A-L show the developmental potential of M phase-iPS cells

FIGS. 3A-F represent the differentiation of embryoid bodies (EB) wasinduced by retinoic acid as described in Material and Methods. EBformation was accompanied by loss of GFP expression.

FIG. 3A represents a differentiating embryoid body.

FIG. 3D represents the GFP expression of the differentiating embryoidbody of FIG. 3A.

FIG. 3B represents differentiated embryoid bodies.

FIG. 3E represents the GFP expression of differentiated embryoid bodiesof FIG. 3B.

FIGS. 3C and F correspond respectively to the higher magnification ofFIGS. 3B and 3E.

FIGS. 3G-I represents chimeric mice produced using M phase iPS cells.Two different M phase iPS clones produced viable chimeras afterinjection into CD1 blastocysts.

FIG. 3J shows the black color of the F1 pups (from the (B6×JF1)genotype) and demonstrates germline transmission.

FIG. 4A-N show the reprogramming of permeabilized MEFs induced by Mphase Xenopus egg extracts.

FIG. 4A represents a curve showing the proliferation rate of M phaseextract-treated MEFs (circles) compared to mock-treated MEFs (squares)at different days (D) after exposure. Error bars represent s.e.m. (n=4).Y-axis represents the total cell number×10⁵.

FIGS. 4B-G represent the morphology of colonies formed followingtreatment of MEFs with M phase Xenopus egg extracts (phase contrast).

FIGS. 4B-E represent respectively different colonies induced by M phaseextract treatment at low magnification (×34).

FIGS. 4F and 4G represent the morphology of colonies at highermagnification.

FIG. 4H-J represent the induction of OCT4 positive colonies followingexposure to M phase egg extracts of wild type MEFs (immunofluorescenceanalysis); Scale bar, 100 μm. FIG. 4J represents the induction of GFPexpression in OCT4-GFP MEFs after incubation with M phase extracts; FIG.4H represent the phase contrast; FIG. 4I represent the DNA labelling(DAPI); scale bar, 50 μm.

FIGS. 4K-M represent the Induction of alkaline phosphatase activity inES cells (FIG. 4K), MEFs (FIG. 4L) and in MEFs after exposure to M phaseegg extracts (FIG. 4M).

FIG. 4 N represents the induction of the expression of pluripotencymarkers (Oct4, Nanog and Rex1, first to thirds column respectively) anddownregulation of Zfpm2 (a differentiation marker; fourth column) afterincubation with M phase egg extracts. Quantitative RT PCR was performedusing M phase extract- and mock-treated MEFs. Error bars represents.e.m. (n=3).

FIGS. 5A-AB show the remodeling of chromatin structure and accelerationof DNA replication in MEF nuclei incubated with M phase Xenopus eggextracts.

FIGS. 5A-D represent the morphology of MEF nuclei incubated for 40 minwith buffer alone (FIG. 5C and FIG. 5D) or with M phase egg extracts(FIG. 5A and FIG. 5B). Nuclei were stained with DAPI (scale bar=10 μm).

FIGS. 5E-G represent the morphology of MEF nuclei treated with bufferalone (FIG. 5E) or M phase egg extracts at 40 min (FIG. 5F) or 60 min(FIG. 5G). Nuclei (stained with DAPI) show different degrees ofchromatin compaction. Scale bar=10 μm.

FIGS. 5H-O represent the phosphorylation of histone H3 at Ser 10(phospho H3) and loss of HP1-α bound to chromatin after exposure of MEFnuclei, or not (Mock), to M phase egg extracts. MEF nuclei were fixedand stained with the corresponding antibodies and DNA was stained withDAPI. Scale bar=10 μm.

FIG. 5H represents the DNA labelling (DAPI) of a mock treated MEFnucleus.

FIG. 5I represents the labelling with an anti phosphorylated histone H3(Ser 10) (phospho H3) antibody of a mock treated MEF nucleus.

FIG. 5J represents the DNA labelling (DAPI) of a mock treated MEFnucleus.

FIG. 5K represents the labelling with an anti HP1-α antibody of a mocktreated MEF nucleus.

FIG. 5L represents the DNA labelling (DAPI) of a M phase extract treatedMEF nucleus.

FIG. 5M represents the labelling with an anti phosphorylated histone H3(Ser 10) (phospho H3) antibody of a M phase extract treated MEF nucleus.

FIG. 5N represents the DNA labelling (DAPI) of a M phase extract treatedMEF nucleus.

FIG. 5O represents the labelling with an anti HP1-α antibody of a Mphase extract treated MEF nucleus.

FIGS. 5P-R represents the analysis of the expression of chromatin-boundphosphorylated histone H3 at Ser 10 (FIG. 5P, upper panel), Lamin B1(FIG. 5Q, upper panel) and HP1-α (FIG. 5R, upper panel) in MEF nucleiafter incubation, or not (A), with M phase egg extracts (B). Chromatinwas collected by centrifugation after treatment as described in Example2. Samples were analyzed by western blotting using the correspondingantibodies. Histone H3 was probed as loading control (FIGS. 5P, 5Q and5R, lower panel.

FIGS. 5S-AB represents the analysis of histone modifications in MEFnuclei after incubation in M phase extracts (B) or not (A). Samples wereanalyzed by western blotting using the corresponding antibodies. HistoneH3 was probed as loading control (lower panel of each of FIGS. 5S-AB).

Upper panel of FIG. 5S represents the blotting with anti acetyl histoneH3 (aCH3) antibody.

Upper panel of FIG. 5T represents the blotting with anti acetyl lysine 9histone H3 (H3K9) antibody.

Upper panel of FIG. 5U represents the blotting with anti acetyl lysine 8histone H4 (H4K8) antibody.

Upper panel of FIG. 5V represents the blotting with anti H3K9me3antibody.

Upper panel of FIG. 5W represents the blotting with anti H3K9me2antibody.

Upper panel of FIG. 5X represents the blotting with anti H4K20me3antibody.

Upper panel of FIG. 5Y represents the blotting with anti H3K4me3antibody.

Upper panel of FIG. 5Z represents the blotting with anti H3K4me2antibody.

Upper panel of FIG. 5AA represents the blotting with anti H3K27me3antibody.

Upper panel of FIG. 5AB represents the blotting with anti histonevariant H3.3 antibody.

FIG. 6: Pre-incubation with M phase Xenopus egg extracts accelerates therate of DNA replication of MEF nuclei in interphasic egg extracts.

FIG. 6A is a schematic representation of the procedure used to evaluateDNA replication in MEF nuclei after incubation with Xenopus M phaseand/or interphase egg extracts.

FIG. 6B represents the DNA replication of permeabilized MEF nuclei (linewith circles) and Xenopus sperm nuclei (line with squares) in Xenopusinterphase egg extracts. The percentage of DNA replication is relativeto the total DNA input in the reaction (see Material and Methods ofExample 2). Y-axis represents the % of DNA replication, and X-axisrepresents the incubation time in min.

FIG. 6C shows that the pre-incubation of permeabilized MEF nuclei in Mphase egg extracts (CSF; line with triangles) enables them to replicateDNA as efficiently as sperm nuclei in interphasic egg extracts (linewith squares). As control, interphasic extract is shown (line withcircles). Y-axis represents the % of DNA replication, and X-axisrepresents the incubation time in min.

FIG. 7: Incubation with M phase Xenopus egg extracts does not affect theviral integration of the OKSM trangenes.

Viral integration of each transgene (Oct4, Sox2, Klf4 and c-Myc) in thedifferent cell populations was assessed by quantitative PCRamplification. The different MEF populations were harvested 21 daysafter infection and their DNA extracted.

First bars (middle grey) correspond to infected, non-permeabilized cells(OKSM);

Second bars (dark grey) correspond to infected, streptolysin-O(SLO)-permeabilized and mock-treated cells (OKSM+SLO+buffer);

Third bars (light grey) corresponds to SLO-permeabilized and M phaseextract-treated cells (OKSM+SLO+M phase extract).

Four independent experiments are shown and errors bars represent s.e.m.(n=4).

Y-axis represents the relative number of integrated transgenes, measuredby Q-PCR.

FIG. 8 represents Scatter plots with computation of the Pearson'scorrelation coefficient (R²) showing the comparisons of global geneexpression between ES cells and MEFs (left) and between ES and M-iPScells (right). Lines indicate the differentially expressed genes betweenpaired cell types.

FIGS. 9A and B represent bisulfite sequencing of DNA from MEFs, ES cellsand M-iPS cells. The amplified regions are indicated by a solid bluebar. Each horizontal row of circles represents the CpG dinucleotides ofan individual molecule. Solid circles depict methylated CpGs, opencircles unmethylated CpGs.

FIG. 9A represents the analysis of the promoter region of Oct4.

FIG. 9B represents the analysis of the promoter region of Nanog.

FIG. 10 represents the down-regulation of the pluripotency markers Oct4,Nanog and Klf4 and up-regulation of the differentiation markers Sox1,Sox7, Sox17 and Brachyury (Brach) upon EB differentiation. The analysiswas performed by quantitative RT-PCR amplification of RNA from ES cells,ES-derived embryoid bodies (EB^(ES)), M-iPS and M-iPS derived embryoidbodies (EB^(M-iPS)) and normalized to the mean expression of Actin, HPRTand GAPDH. Histograms represent the ratio between the correspondingembryoid bodies and pluripotent cells (ES, blue bars or M-iPS cells, redbars) and their. Error bars represent s.e.m. (n=3). Y-axis representsthe fold induction of mRNA relative to housekeeping genes.

FIGS. 11A-C: the incubation with M phase Xenopus egg extracts does notdemethylate DNA of MEF nuclei

Bisulfite sequencing was performed in mock-treated and M-phase treatedMEF nuclei and ES cells. Amplified regions are indicated by a solid bluebar. Each horizontal row of circles represents the CpG dinucleotides ofan individual molecule. Solid circles depict methylated CpGs, opencircles unmethylated CpGs. The parental allele origin (M: maternal; P:paternal) was determined in MEFs and iPS cells by using DNApolymorphisms between C57BL/6J and JF1 backgrounds. Blue triangles showindividual CpGs that are absent due to polymorphisms.

FIG. 11A represents the analysis of the promoter region of Oct4.

FIG. 11B represents the analysis of the promoter region of Nanog.

FIG. 11C represents the analysis of the promoter/imprinting controlregion of the imprinted Snrpn gene.

FIG. 12: Stability of the blockage of the isolated extract of femalegerminal cells in metaphase.

Y-axis represents the % of DNA synthesis, and X-axis represents theincubation time in min.

Capacity of the isolated extract blocked in phase M to synthesize DNAwith Ca2+ (diamonds), or without Ca2+ (square), is measured byincorporation of [³²P]αdCTP.

When the extract is blocked in metaphase, it does not synthesize DNAwithout Ca2+, but it synthesizes DNA in presence of Ca2+.

EXAMPLES Example 1 Extract Preparation

Mitotic extracts are prepared through a procedure similar to that usedfor interphasic extract. Eggs should not be activated, however, and EGTAshould be added to buffers to chelate traces of calcium, either presentin solutions or released from intracellular stores.

1. Set the centrifuge at 1° C. Cool all tubes, adaptators, and syringesto 4° C. before starting the preparation of the extract.

2. Transfer the eggs to a glass beaker and rinse with HSB (HSB-CSF: 15mM Hepes pH 7.6; 110 mM NaCl; 2 mM KCl; 1 mM MgSO4; 0.5 mM Na2HPO4; 2 mMNaHCO3+2 mM EGTA). It is advantageous to pool the eggs from the samefrogs.

3. Add distilled water and leave the external jelly coat to swell for 5min at room temperature.

4. Add HSB-CSF 0.3×, cysteine 2%, pH 7.9 (the solution should be usedwithin 6 hrs of preparation), and dejelly by gentle swirling atintervals. This takes 5 to 10 min and complete removal of the jelly isobtained when the eggs can be tightly packed together, slightlydeformed. It is important to obtain a complete dejellification. At thisstage, success depends on both the rapidity with which the preparationis done and the strict observation of the cold temperature conditionsafter step 10.

5. Rinse immediately at least 5 times with 100-200 ml HSB-CSF per ml ofeggs. If at this point necrosis is visible in more than 20% of the eggs,do not proceed further. Transfer to 50-ml glass beaker.

6. Transfer the eggs in a large glass Petri dish for observation under amicroscope. Eggs should not show any signs of spontaneous activation.

7. Transfer to a cold Ultra-clear tube. Rinse with cold XB-CSF (10 mMHEPES pH 7.7; 100 mM KCl; 1 mM MgCl2; 5% Sucrose; 1 mM DTT; 5 mM EGTA)containing 10 μg/ml protease inhibitors and 100 μg/ml cytochalasin B.Use 1 ml for 3 ml eggs.

8. Leave the tube in ice for 5 to 10 min to chill the eggs.

13. Remove the excess buffer and pack the eggs by centrifugation at 150g, 45 sec, 1° C., in a Sorvall swinging rotor or equivalent.

14. Rapidly remove the excess buffer and centrifuge at 17,000 g (SorvallHB4 swinging rotor, 10K), for 10 min at 1° C. The centrifugation crushesthe eggs and the soluble content is thus exuded.

15. Withdraw the extract by puncturing the side of the tube with a20-gauge needle inserted into a 1 to 5 ml syringe, depending on theamount of soluble extract. Insert the needle just above the blackpigment layer and collect the cytoplasmic layer, avoiding the yellowlipid top layer. Transfer to a cold Ultra-clear tube. Add 10 μg/mlprotease inhibitors, 10 μg/ml cytochalasin B, 1/20 volume Energy Mix 20×(Energy Mix—CSF 20×: 200 μg/ml Creatine Kinase; 200 mM CreatinePhosphate; 20 mM ATP; 20 mM MgCl2; +2 mM EGTA), and 5% glycerol. Mixgently.

16. Centrifuge again in the same conditions.

17. Collect the supernatant in a cold tube. If necessary, add 200 μg/mlcycloheximide to prevent protein synthesis. Store at −80° C. in 100 or200 μl aliquots previously frozen in liquid nitrogen. Proteinconcentration in low speed extracts is around 50 mg/ml and RNAconcentration, mainly in ribosomes, is 5-10 mg/ml. Aliquots should beused only once and should not be frozen again after thawing.

Example 2 Synergic Induction of Pluripotent Cells by Combined Exposureto Mitotic Egg Extracts and Transcription Factors

Introduction

Nuclear transfer (NT) experiments in frogs and then in mammalian eggshave demonstrated that somatic cells can be reprogrammed to pluripotency(1-4). More recently, induction of pluripotency in somatic cells byectopic expression of the four transcription factors Oct4, Klf4, Sox2and c-Myc (OKSM) has been used to produce induced pluripotent stem (iPS)cells, which are highly similar to embryonic stem (ES) cells. Notably,murine iPS cells have a complete developmental potential as demonstratedby their capacity to form teratomas, generate chimeras and contribute tothe germline. However, the efficiencies of both iPS cell production andNT remain low and most of the obtained reprogrammed cells appear to beonly partially reprogrammed. The epigenetic memory of the cell is onekey barrier, which has to be overcome to efficiently reprogramdifferentiated cells (5). Thus, additional factors may be needed toimprove reprogramming efficiency (6, 7) and many efforts have been doneover the last years to optimize these procedures. It has been suggestedthat different reprogramming strategies could be associated to synergizetheir efficiencies (8). Several attempts have been made by usingcellular extracts to reprogram somatic cells, but they failed toreproduce the range of effects obtained by NT.

In NT experiments, reprogramming is induced by exposure of transplantednuclei to the cytoplasm of the receiving oocyte. However, NTreprogramming appears hard to study in vitro due to the difficulty toobtain large quantities of mammalian oocytes. Xenopus eggs, which can beobtained in large amounts, can remodel the nuclear lamina of reversiblypermeabilized mammalian cells (9) and Xenopus egg extracts canup-regulate Oct4 expression in cells that already express Oct4 (10),similarly to what observed when adult mouse nuclei are injected inXenopus oocytes (11). More recently, it was reported that thereplication origin pattern and chromosome organization of Xenopuserythrocyte nuclei could be remodeled by metaphase-arrested extracts (Mphase extracts) from Xenopus eggs (12). The Inventors furtherinvestigated whether pre-incubation of mouse embryonic fibroblasts(MEFs) with Xenopus egg extracts could increase the efficiency of NT andiPS production. The Inventors show that M phase, but not interphase,Xenopus egg extracts increased NT efficiency and engaged MEFs into astem cell program. They also induced a global change of MEF chromatinstructure and replication properties. In particular, M phase extractsreset the level of several epigenetic marks in MEF nuclei, independentlyof their role in chromatin activation. Moreover, M phase extracts, butnot interphase extracts, partially reprogrammed permeabilized MEFs toform colonies, which expressed pluripotency markers. Finally, iPS cellinduction by ectopic expression of OSKM was 45-fold increased when MEFswere incubated in M phase Xenopus egg extracts. The resulting iPS cellswere fully reprogrammed, as shown by their capacity to produce chimerasand colonize the germline.

Results

Pre-Treatment with M Phase Xenopus Egg Extracts Improves Efficiency ofBoth Nuclear Transfer and iPS Cell Production in Mammals

The Inventors first asked whether M phase Xenopus egg extracts couldimprove the highly inefficient NT of MEFs (13). Permeabilized MEF nucleiin G1 phase were pre-incubated with M phase (FIG. 1A) or interphasicXenopus egg extracts or buffer alone and their progression to blastocyststage, after NT, was compared. NT of G1 MEFs nuclei led to 11%blastocysts (FIG. 1B and Table 1), a value that was significantly lowerthan what obtained after NT of metaphase ES nuclei (55%), which werepreviously described as the best donor nuclei for NT (14). ConditioningMEF nuclei in M phase egg extracts significantly increased the rate ofblastocyst formation to a level comparable to that obtained withmetaphase ES nuclei (45%) (FIG. 1B and Table 1). These data show that Mphase Xenopus egg extracts efficiently improve reprogramming of somaticcells by NT. Conversely, pre-incubation with interphasic egg extractsdid not improve but rather slightly decreased NT efficiency (3%),indicating the importance of the mitotic state of the reprogrammingextract. Since both mitotic MEFs and G1 ES nuclei were relativelyinefficient donors for NT in metaphase-blocked oocytes (summarized inTable 1), in Inventor's results also suggest that treatment with M phaseXenopus extracts can remodel MEF nuclei toward both a mitotic andpluripotent state.

The Inventors then checked whether treatment with M phase Xenopus eggextracts could also improve the efficiency of iPS cell production. Thegeneration of iPS cells by viral-mediated expression of the OSKMtranscription factors in mouse and human cells, although with lowefficiency, was a breakthrough in reprogramming of somatic cells to apluripotency state (15-19). The Inventors therefore combined OSKMover-expression and incubation with M phase Xenopus egg extracts (M-iPScells) using the experimental strategy shown in FIG. 1C. OCT4-GFP MEFswere infected with retroviruses encoding the four transcription factors,permeabilized, incubated with M phase extracts and then resealed ontogelatine-coated plates in ES medium. The Inventors checked byquantitative PCR that the M phase extract treatment did not influencethe viral integration of the OKSM transgenes (FIG. 7). Seven days afterinfection, the Inventors determined the proportion of OCT4-GFP positivecolonies, which is related to full reprogramming events since endogenousOCT4 re-expression has been reported to be a stringent reporter ofreprogramming (2). The number of GFP positive colonies was 45-foldhigher in OSKM-induced cells exposed to M phase egg extracts (M-iPScells) than in cells that over-expressed only OSKM, with or withouttreatment with streptolysin-O (SLO) (FIG. 1D). Thus, a short incubationof mammalian somatic cells in M phase Xenopus egg extracts greatlyincreases the yield of fully reprogrammed iPS cells.

TABLE 1 represents the in vitro development of embryos obtained usingMEF nuclei exposed to M phase Xenopus egg extracts and injected intoenucleated mouse oocytes recon- % of 2-cell embryos structed activated2-cell 4/8-cell morula blastocyst Mitotic ES 175 154 87% ND 55% cells(134) (84) G ES cells*  39  36  28 ND 28% 11% (Zhou et al. (8)  (3) 2001 G1 MEFs 263 195 140 56% 27% 11% (79)  (38) (16) Mitotic ND ND ND NDND  6% MEFs** (Li et al. 2003) MEFs + 178  84  30 27%  7%  3%interphasic (8)  (2)  (1)  extracts MEFs + M 148  87  49 78% 67% 45%phase extracts (38)  (33) (22) Percentage of embryos relative to 2-cellembryos obtained after nuclear transfer of ES nuclei and MEF nuclei thathad been pre-incubated in mock buffer, interphase or M phase Xenopus eggextracts. Mitotic and G1 ES nuclei were isolated and injected aspreviously described (33) and mitotic MEF nuclei as in Li et al. (13).*refers to sv129/sv cell line; **refers to 129/Svpas cell line.

Characterization of M-iPS Cells

M-iPS cells presented an ES-like morphology and uniform expression ofthe pluripotency markers alkaline phosphatase, OCT4, NANOG, and SSEA1(FIGS. 2A-S). Moreover, the levels of expression of differentpluripotency markers were measured by quantitative PCR and were similarto those in ES cells (FIGS. 2T-U). The transcriptomic profile of M-iPScells, MEFs and ES cells were analyzed (FIG. 7) and scatter plots of DNAmicroarrays analyses confirmed the similarity between M-iPS and ES cells(R²=0.9175). Efficient reprogramming has been tightly linked tohypo-methylation of DNA on promoters of key regulators of pluripotency,such as Oct4 and Nanog [Maherali N, et al. Cell Stem Cell. 2007 Jun. 7;1(1): 55-70]. The DNA methylation profiles of M-iPS cells and ES cellswere similar (FIGS. 8A-B), confirming the efficiency of reprogrammingobtained by combining M phase Xenopus egg extracts and OKSM expression.

The Inventors then investigated the ability of M-iPS clones todifferentiate. When induced to differentiate, all tested M-iPS clonesformed embryoid bodies (FIG. 3A-F) and the stem cell markers Oct4, Nanogand Klf4 were down-regulated (FIG. 10), whereas markers ofdifferentiation in the three germ layers were up-regulated with levelscomparable to those observed in embryoid bodies obtained from ES cells(FIG. 10) and (20-23).

Finally, the complete reprogramming of the M-iPS clones was demonstratedin vivo by the capacity of two different clones, one male and onefemale, to produce adult chimeras after injection into CD1 blastocysts(FIGS. 3G-I and Table 2). In addition, germline transmission was alsosuccessful as shown by the production of F1 black offspring (due to theB6×JF1 genetic background) after mating these chimeras with CD1 albinoanimals (FIG. 3J).

The Inventors conclude that M phase Xenopus egg extracts have a strongpositive effect on the efficiency of iPS cell production. Importantly,this action is not additional but synergistic, since the reprogrammingefficiency (number of GFP-positive colonies, see FIG. 1D) when the twostrategies are combined is much higher than the simple addition of theirrespective efficiency.

TABLE 2 represents the developmental potential of iPS cells derived fromMEFs exposed to M phase Xenopus egg extracts (M phase iPS cells).Injected Number blasto- Born of % of Female Male cysts embryos chimeraschimeras chimeras chimeras Clone 233 71 31 46% 25 6 #1 (male) Clone 8510 7 70% 3 4 #2 (female) Percentage of chimeras obtained after injectionof two different clones of M phase iPS cells (one male and one female)into CD1 blastocysts and analysis of their ability to colonize thegermline.

Xenopus M Phase Egg Extracts Partially Reprogram Mammalian Fibroblasts

To characterize the synergistic effect of M phase Xenopus egg extracts,the Inventors first asked whether this treatment alone could modify thelimited proliferation potential of MEFs (24). Treatment with M phase eggextracts strongly increased the proliferation rate of MEFs during atleast two cell cycles (FIG. 4A) and induced also the formation of a fewcolonies that expanded over a few days before growth arrest (FIG. 4B-G).These colonies were less numerous than upon M-iPS cell induction andwere never seen in mock-treated MEFs.

Growth stimulation was also accompanied by expression of pluripotencycell markers, which were never observed in mock-treated cells. Indeed,alkaline phosphatase expression (a marker of partial reprogramming) wasinduced upon M phase treatment (FIGS. 4K-M) and endogenous expression ofOCT4, a more stringent marker of pluripotency (2), was detected incolonies by immuno fluorescence as well as GFP expression driven by theOct4 promoter (FIGS. 4H-J). Interestingly, alkaline phosphatase wasexpressed in a relatively high proportion of M phase extract-treatedcells, including those that did not progress further to form colonies(FIGS. 4K-M). The presence in several independent experiments of clonesthat expressed OCT4, or alkaline phosphatase, or both suggests that Mphase egg extracts favor the development of a heterogeneous cellpopulation with different levels of reprogramming. This is in agreementwith the heterogeneity observed during the production of iPS cells byusing OSKM over-expression and it is likely to be the result of astochastic process (25). These results indicate that M phase extractsalone can change the cell cycle properties and can induce a partial andtransient reprogramming of MEFs.

Seven days after treatment with M phase egg extracts, the expression ofthe pluripotency markers Oct4, Nanog and Rex1 was confirmed byquantitative RT PCR (FIG. 4E) in whole unselected cell populations, aspluripotency markers were often detected before colony formation.Primers used for Q-PCR analyses were specific for mouse transcripts andthey could not amplify RNA from M phase Xenopus extracts, confirming theinduction of expression of the endogenous mouse genes. In addition,Zfpm2, a transcription factor expressed in MEFs but not in ES cells(18), was down-regulated after exposure to M phase egg extracts (FIG.4N).

Overall, the Inventors' data suggest that M phase Xenopus egg extractsalone are sufficient to partly reprogram MEFs, as indicated by theup-regulation of pluripotency genes and down-regulation of genesnormally expressed in MEFs and the rapid but transient induction ofproliferation. Neither of these effects was observed when usinginterphase Xenopus egg extracts, in agreement with the previouslyreported failure to reprogram cells using Xenopus egg extracts describedin (26).

Treatment with M-Phase Xenopus Egg Extracts Induces Mitotic Features andModifies the Global Epigenetic Signature

The observations that only M phase and not interphasic Xenopus eggextracts had a reprogramming effect on reversely permeabilized MEFs aswell as on NT efficiency indicate that the mitotic stage of the donorextract is crucial. Therefore, the inventors investigated whetherexposure of MEFs at the G1 phase to M phase egg extracts could inducemitotic markers in the reprogrammed nuclei. Indeed, exposure to M phaseXenopus egg extracts drove MEF nuclei into a mitotic-like stage,accompanied by modification of the chromatin structure (FIGS. 5A-D)followed by global condensation, as shown by the formation of condensedchromatin fibers (FIGS. 5E-G). MEF nuclei exposed to M phase eggextracts also showed phosphorylation of histone H3 on Ser 10, anddissociation of the nuclear envelope component Lamin B1 (27, 28), afactor involved in the nuclear structure (FIGS. 5H-O and 5R), alldistinctive features of entry in mitotic phase.

Exposure to M phase egg extracts also appeared to erase the chromatinsuperstructure organization, as revealed by the loss of heterochromatinfoci visualized by DAPI staining together with the loss of HP1expression (FIGS. 5A-R). The Inventors thus further investigated whetherM phase egg extracts modified the global epigenetic signature of MEFnuclei. The Inventors first determined the level of histone acetylationbecause it has been shown that the histones of the donor nuclei aredeacetylated during NT (29, 30). Western blots analysis showed thatincubation of MEF nuclei with M phase Xenopus egg extracts reduced thelevel of acetylation of H3 (particularly H3K9) and of H4 at Lysine 8(FIGS. 5S-AB).

The Inventors then asked whether the Xenopus egg extracts could alsomodify the histone methylation profiles, as histone hypomethylation hasbeen correlated with the epigenetic plasticity of somatic mammaliancells (31). A short incubation of MEF nuclei with M phase Xenopus eggextracts globally reduced the level of H3K9me2-me3, H4K20me3 andH3K4me2-me3 as shown by western blotting (FIGS. 5S-AB). Conversely, thelevel of H3K27me3 did not change upon incubation with M phase extracts,suggesting that this mark is more stable. The global demethylation atH3K9 might contribute to the improvement of NT efficiency followingincubation with M phase egg extracts because maintenance of H3K9tri-methylation has been associated with developmental failure during NT(32). Altogether, these results show that incubation with M phaseXenopus egg extracts broadly modifies the epigenetic signature ofmammalian somatic nuclei by resetting several, but not all, epigeneticmarks.

Moreover, incubation with M phase Xenopus egg extracts also induced areduction of the global level of the histone variant H3.3, which hasbeen recently implicated in cell identity memory during reprogramming byNT (33) (FIGS. 5S-AB).

Finally, the Inventors analyzed the DNA methylation profile, another keymarker of cell memory. Bisulfite sequencing was performed and showedthat incubation in M phase Xenopus egg extracts for 40 minutes did notmodify the DNA methylation status of the pluripotency genes Oct4 andNanog (FIGS. 11A-C).

In summary, Xenopus M phase extracts drive MEF nuclei into a mitoticstate and also remodel their chromatin structure. These results couldexplain the strong synergistic effect of the treatment with M phaseXenopus extract on NT and iPS cells production.

MEF Nuclei are Adapted to an Embryonic Replication Program whenPre-Incubated in M Phase Xenopus Egg Extracts.

The Inventors previously showed that M phase Xenopus egg extracts couldreset the replication program of nuclei from differentiated Xenopuscells and allow the transition from a somatic to an embryonic profile ofDNA replication (12). The Inventors thus asked whether MEF nuclei couldbe similarly reprogrammed. To this aim nuclei from MEFs synchronized inG1 were incubated either with interphasic Xenopus egg extracts or firstexposed to M-phase egg extracts before transfer into interphasic eggextracts and then their ability to replicate DNA was assessed (FIG. 6A).Nuclei exposed only to interphase egg extracts did not (or very poorly)replicate DNA (FIG. 6B). Conversely, pre-incubation of MEF nuclei in Mphase egg extracts induced DNA replication with a kinetic nearly similarto that of Xenopus sperm nuclei when further transferred to aninterphase extract (FIG. 6C). The Inventors conclude that mouse somaticnuclei passing through mitosis in Xenopus egg extracts are partiallyreprogrammed and acquire the accelerated rate of DNA replicationcharacteristic of Xenopus early embryos.

Discussion

Reprogramming of Mouse Embryonic Fibroblasts by Xenopus Egg Extracts

The experiments described here show that a short incubation of mammaliansomatic nuclei or cells with M phase Xenopus egg extracts improves theefficiency of both NT and iPS cell production. This suggests theexistence of common barriers limiting the efficiency of reprogramming byNT and iPS cells that pre-incubation in M phase Xenopus egg extractmight help removing these barriers. Moreover, the results presented herealso emphasize that combining different strategies can improve thereprogramming of mammalian somatic cell nuclei. Neither NT norheterocaryons can be used in combination with iPS cells due to technicallimitations. However, Xenopus egg extracts can be obtained in largeamount and can be used to increase the yields of iPS cells.

The Inventors show that incubation with M phase Xenopus egg extracts issufficient to improve the efficiency of NT using MEF nuclei up to thelevel observed with pluripotent ES cells. Furthermore, reversiblypermeabilized MEFs incubated in M phase Xenopus egg extracts acquireseveral features of pluripotent cells, such as induction of cellproliferation, formation of colonies, expression of ES cell markers,including the expression of OCT4, one of the most stringent marker ofpluripotency (34). This reprogramming activity is not stable; coloniesstop growing after a couple of rapid cell cycles. However, this partialreprogramming activity is enough to increase by 45-fold the productionof fully reprogrammed iPS cells by viral transduction of OKSM. Thissynergic effect is probably underestimated since the proportion ofefficiently permeabilized MEFs does not exceed 30% in the Inventors'hands. The resulting M-iPS clones appear to be well reprogrammed sincethe obtained clones could efficiently produce chimeras and colonize thegermline. This synergic effect suggest that incubation in Xenopus eggextracts can induce modifications of the genome features of somaticmammalian cells, thus opening a larger window of action forreprogramming by NT or OKSM expression.

Importance of Exposure to Mitotic/Meiotic Conditions for ReconditioningDifferentiated Nuclei

The Inventors' experiments show that the mitotic state of the Xenopusegg extracts is crucial. Xenopus interphasic egg extracts neitherinduced reprogramming in permeabilized MEFs nor improved NT efficiency.Conversely, M phase Xenopus egg extracts induced a global mitoticsignature in G1 MEF nuclei, as revealed by the phosphorylation ofhistone H3 on Ser 10 and remodeling of the nuclear structure. Thisglobal reorganization of chromatin at mitosis is likely to be criticalfor the reprogramming activity of M phase Xenopus egg extracts.Transition through mitosis has always been found to be crucial in NTexperiments performed in the mouse, where zygotes temporally arrested inmitosis support nuclear reprogramming much more efficiently thatinterphase zygotes (35). Altogether, these results indicate thatefficient reprogramming requires not only an early embryonic pluripotentcontext, but also transition through mitosis.

Incubation of donor somatic nuclei in mitotic egg extracts could helpresynchronizing the cell cycle of donor nuclei to make them compatiblewith an early development context. The Inventors show that MEF nuclei,like Xenopus somatic cell nuclei but differently from sperm nuclei, arenot competent to replicate their genome in interphasic Xenopus eggextracts. The requirement of a mitotic reprogramming phase may explainwhy, in NT experiments, nuclei from half-cleaved embryos develop muchbetter than nuclei from normal blastulae (36). Indeed, such nuclei werederived from embryos that failed to divide during the 1^(st) cleavage,implying that they should have gone through a mitotic stage beforeentering in S phase. In mouse, inefficient development occurs whennuclei are transferred into pre-activated oocytes, whereas the bestdevelopmental rates are observed when activation occurs 1-3 hours afternuclei transfer (37). The Inventors' observations provide an explanationto these data by showing that mitotic, but not interphasic Xenopus eggextracts can reprogram differentiated cells.

M Phase Xenopus Egg Extracts Remodel the Global Organization of SomaticMammalian Genomes

In addition to the cell cycle synchronization effects, conditioningnuclei in a mitotic embryonic context may facilitate reprogramming ofgene expression. During mitosis, most pre-existing transcription andreplication factors are erased from chromatin (38). For instance, TBP,the main component of the transcription machinery which is required fortranscription by all three polymerases, as well as TFIIB are removedfrom the chromatin of somatic cell nuclei incubated in egg extracts,together with the disappearance of the nucleoli (39). The Inventors'experiments show that M phase Xenopus egg extracts efficiently induce aglobal mitotic signature in G1 MEF nuclei, as revealed by the loss ofHP1, phosphorylation of histone H3 on Ser 10 and remodeling of thenuclear structure. Interestingly, marks associated with transcriptionalrepression (H3K9me2, H3K9me3, H4K20me3) and with active chromatin(acetyl H4K8, acetyl H3K9, H3K4me3, H3K4me2) are both reduced inchromatin of MEF nuclei incubated with M phase extracts. This event isreminiscent of the atypical bivalent epigenetic signature of ES cells(40) and could promote reprogramming by resetting the memory of thesomatic nuclei. Histone demethylation also appears to be an interestingfeature of the action of the M phase Xenopus egg extracts. However, thereduction of epigenetic marks is not complete, suggesting that somedefined nuclear structures could remain after incubation with M phaseextracts.

The Inventors' results show that pre-incubation with M phase Xenopus eggextracts can recapitulate reprogramming events occurring during NT.Indeed, they explain the global epigenetic modifications that have beendescribed during reprogramming of mammalian somatic nuclei injected innon-activated, metaphase II mammalian oocytes (29, 30, 41). Thus,Xenopus egg extracts could provide a powerful tool to biochemicallystudy molecular events occurring during NT.

The global reorganization of chromatin at mitosis is likely to becrucial for the reprogramming activity by M phase Xenopus egg extracts.These extracts have the advantage of providing all the genetic andepigenetic factors involved in mitosis as well as in pluripotency, asopposed to reprogramming through ectopic expression of a few genes. Thecombination of both methods leads to a strong synergistic effect,demonstrating the evolutionary conservation of reprogramming circuits.

Material and Methods

Cells and Media

MEFs were derived from 13.5E wild type mouse embryos or fromC57BL/6J-JF1 embryos hemizygous for the OCT4-GFP transgenic allele.Gonads, internal organs and heads were removed before MEF isolation.MEFs were then expanded in high-glucose DMEM (Invitrogen) supplementedwith 10% ES-tested fetal bovine serum (cat N° S1810, Biowest), 2 mML-glutamine (Invitrogen), 1 mM sodium pyruvate (Sigma). MEFs were usedup to passage 5. OCT4-GFP mice were initially created by Pr. Schöler(42) and obtained from Pr. Surani (Wellcome Trust/Cancer Research UKGurdon Institute, Cambridge). The ES cell line CGR8 was obtained from DrC. Crozet (Institut de Génétique Humaine, Montpellier). ES cells weregrown on 0.1% gelatin without feeders. They were cultured at 37° C. in5% CO₂ in ES medium: GMEM supplemented with 10% fetal calf serum, 0.1 mMβ-mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids(Gibco), 2 mM L-glutamine, in the presence of 1000 U/ml LIF (ES-GRO).

Xenopus Egg Extract Preparation and Replication Reactions

Xenopus mitotic and interphasic egg extracts as well as demembranatedsperm nuclei were prepared and used as described in Lemaitre et al.(12), Menut et al. (43) and the detailed protocol available atwww.igh.cnrs.fr/equip/mechali/. MEF nuclei were prepared from confluentMEFs at early passages (up to P5) following the procedure described forXenopus erythrocyte nuclei (12). Briefly, MEFs were trypsinized andwashed twice with PBS. MEFs were incubated in hypotonic buffer (10 mMKHEPES pH7.5; 2 mM KCl; 1 mM DTT; 2 mM MgCl₂; 1 mM PMSF; proteaseinhibitors) for 1 hour. Swelled cells were then homogenized with 20 to30 strokes and then incubated in hypotonic buffer containing 0.2% TritonX-100 on ice for 3 minutes. Nuclei were washed twice in isotonic buffer(10 mM KHEPES, 25 mM KCl, 2 mM MgCl₂, 75 mM sucrose and proteaseinhibitors). Nuclei were finally centrifuged through a 0.7M sucrosecushion and resuspended in isotonic buffer supplemented with 20%sucrose. Sperm nuclei and MEF nuclei (1000 nuclei/μl and 500 nuclei/μlrespectively) were incubated in S phase or M phase (CSF) extracts. DNAsynthesis was measured by [³²P]αdCTP incorporation in Xenopusinterphasic egg extracts as previously described (43). Nuclei transferfrom M phase extracts to interphasic extracts was performed as describedpreviously (12).

Streptolysin-O Permeabilization and M Phase Extract Treatment

MEFs were permeabilized with streptolysin-O (SLO) mainly as described byTaranger et al. (44). Briefly, MEFs were trypsinized, washed twice inPBS and then resuspended in cold Ca²+ and Mg²⁺-free Hanks' Balanced SaltSolution (HBSS) at 1000 cells/μl with 250 ng/μl SLO (Sigma S0149). Cellswere incubated at 37° C. with gentle agitation for 50 min and thenwashed twice with ice cold HBSS. Permeabilized cells were incubated in Mphase Xenopus egg extracts or buffer (1000 cells/μl of extracts) for 40min, washed twice in HBSS and resealed on gelatin in complete ES mediumsupplemented with 2 mM CaCl₂ for 2 hours and then cultured in completeES medium.

M Phase-Extract Treated iPS Cells Production

Constructs in pMXs retroviral vectors encoding Oct4, Sox2, Klf4 andc-Myc (obtained from Addgene) were transfected in Platinum HEK cellsusing the Lipofectamine 2000 transfection reagent (Invitrogen),according to the manufacturer's recommendations. 30 μl of Lipofectamine2000 were added to 750 μl OPTIMEM and mixed with 12 μg DNA that had beendiluted into 750 μl OPTIMEM and incubated for 5 min. After 20 minincubation at 20° C., the DNA/Lipofectamine 2000 mixture was added dropby drop to Platinum HEK cells. 48 h after transfection, supernatantswere collected, filtered through 0.45 μm Millex-HV (Millipore) filtersand supplemented with 12 μg/ml polybrene. OCT4-GFP MEFs were seeded on0.1% gelatin at a density of 8.10⁵ cells in 56 cm² Petri dishes and thefour virus containing supernatants were pooled in equal amounts andadded to the MEFs. 18 hours later, supernatants were removed and cellscultured in complete ES medium. Five-six hours later, cells weretrypsinized and permeabilized with SLO as described above and thenincubated either in mock buffer (HBSS) or in Xenopus M phase eggextracts for 40 min. After treatment, cells were washed twice and plated(8.10⁵ cells per 56 cm²) in gelatin-covered dishes with ES mediumsupplemented with 2 mM CaCl₂. After 2 hours, medium was removed andreplaced by complete ES medium until appearance of OCT4-GFP positivecolonies. M phase extract-treated OCT4-GFP positive colonies weremechanically isolated, individual cells dissociated and plated ontofeeders for analysis that was performed after at least 15 passages onfeeders.

Nuclear Transfer

Nuclear transfer experiments were performed mainly as described in Zhouet al. (45). Briefly, permeabilized MEF nuclei from confluent (B6×129)MEFs were freshly prepared as described above and either directlyinjected into enucleated, metaphase II mouse oocytes or pre-incubated inM phase or interphasic Xenopus egg extracts for 40 min. Beforeinjection, pre-incubated nuclei were washed twice in M16 medium toeliminate the Xenopus egg extract. Before injection, the efficiency oftreatment and chromatin integrity were assessed by visually inspectingthe nuclei with a phase contrast microscope. (B6×129) metaphase ES cellswere isolated as described in Zhou et al. (45).

Differentiation of ES Cells or M Phase Extract-Treated iPS Cells.

ES cells or M phase extract-treated iPS cells were dissociated intosingle cell suspensions with 0.05% trypsin/EDTA and plated at lowdensity in non-adherent bacterial Petri dishes with standard ES culturemedium (without LIF). After 2 days, medium was replaced with ES culturemedium supplemented with 0.5 μM retinoic acid to induce differentiationof embryoid bodies.

Reprogramming Efficiency

Reprogramming efficiency after M-phase extracts treatment was analyzedseven days after infection. The number of OCT4-GFP positive coloniesinduced by the different treatments was counted under a fluorescentmicroscope and compared with the number of colonies obtained fromnon-permeabilized OKSM-infected MEFs from the same infection experiment.Alkaline phosphatase staining was performed using the AlkalinePhosphatase Detection Kit from Sigma Diagnostics according to themanufacturer's procedure. For immunofluorescence, cells in culture werewashed once in PBS and then fixed in 3% paraformaldehyde at roomtemperature (RT) for 15 minutes, washed with PBS and permeabilized withPBS/0.2% Triton X-100 for 5 min. Cells were then washed three times inPBS with 2% BSA for 10 minutes, incubated with anti-OCT-3/-4 (C-10)(Santa-Cruz, sc-5279), anti-NANOG (Abcam, ab21603) or anti-SSEA1 (clone16MC480) (Abcam, ab16285) antibodies for 1 hr and then with thesecondary antibody for 1 h after 3 washes in PBS. DNA was stained withDAPI. Immunofluorescence analysis of M phase extract- or mock-treatedMEF nuclei was performed by spinning the treated nuclei onto coverslipsby centrifugation at 100 g after having been 10-fold diluted in XBbuffer (XB: 100 mM KCl, 0.1 mM CaCl₂, 1 mM MgCl₂, 10 mM KOH-HEPES [pH7.7], 50 mM sucrose supplemented with protease inhibitors) as describedpreviously (43).

Quantitative Reverse Transcriptase (RT)-PCR Analysis

For transcriptional analysis, total RNA was isolated from whole cellpopulations using the RNAeasy Mini Kit and RT was performed using theHigh Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Quantitative PCR was performed on a Lightcycler 480 apparatus using theLightcycler 480 SYBR Green I Master kit from Roche. Quantification datawere normalized to the average expression of the endogenous Hprt1/Gapdhand β-Actin genes within the log-linear phase of the amplification curveobtained for each primer set using the ΔΔCt method. All samples wereprepared in 2 to 3 biological repeats.

Primers for Quantitative RT-PCR: Oct4 (SEQ ID NO: 1)Fw: ttctggcgccggttacagaaccatactcga  (SEQ ID NO: 2)Rev: gaggaagccgacaacaatgagaaccttcag  Rex 1 (SEQ ID NO: 3)Fw: cagctcctgcacacagaaga  (SEQ ID NO: 4) Rev: actgatccgcaaacacctg  Nanog(SEQ ID NO: 5) Fw: ttcttgcttacaagggtctgc  (SEQ ID NO: 6)Rev: agaggaagggcgaggaga  Zfpm2 (SEQ ID NO: 7) Fw: gcgaagacgtggagttcttt (SEQ ID NO: 8) Rev: ggctgtccccatctgattc  β-Actin (SEQ ID NO: 9)Fw: gccggcttacactgcgcttctt  (SEQ ID NO: 10) Rev: ttctggcccatgcccaccat Gapdh (SEQ ID NO: 11) Fw: tggcaaagtggagattgttgc  (SEQ ID NO: 12)Rev: aagatggtgatgggcttcccg  Hprt1 (SEQ ID NO: 13)Fw: tcctcctcagaccgcttt  (SEQ ID NO: 14) Rev: cctggttcatcgctaatc  Sox1(SEQ ID NO: 15) Fw: gtgacatctgcccccatc  (SEQ ID NO: 16)Rev: gaggccagtctggtgtcag  Sox17 (SEQ ID NO: 17)Fw: ctttatggtgtgggccaaag  (SEQ ID NO: 18) Rev: ggtcaacgccttccaagact Sox7 (SEQ ID NO: 19) Fw: gcggagctcagcaagatg  (SEQ ID NO: 20)Rev: gggtctcttctgggacagtg  Brachyury (SEQ ID NO: 21)Fw: cagcccacctactggctcta  (SEQ ID NO: 22) Rev: gagcctggggtgatggta  Klf4(SEQ ID NO: 23) Fw: gagttcctcacgccaacg  (SEQ ID NO: 24)Rev: cgggaagggagaagacact 

DNA Microarrays Analysis

Total double strand cDNAs from ES cells, MEFs and M-iPS cells washybridized on Nimblegen mouse expression 135K arrays and results wereanalyzed with the free trial Arraystar software. Normalization wascalculated with the RMA algorithm (46) implemented in Bioconductor. Theexperiments were performed in triplicates.

Gene-by-gene tests for differential expression between paired cell typeswere performed using a moderated t-statistic (47). P-values wereadjusted using the procedure of Benjamini and Hochberg for controllingthe False Discovery Rate (FDR) (48). Differentially expressed genesbetween the paired cell types were identified using adjusted p valuesbelow 1%.

Bisulfite Sequencing

DNA extraction and bisulfite sequencing of mock-treated and M-phasetreated MEF nuclei, M-iPS cells and CGR8 ES cells were performed aspreviously described (49). Before DNA extraction, GFP positive M-iPScells were sorted with a Facsaria cytometer to avoid contamination byfeeder cells. DNA polymorphisms between the C57BL/6J and JF1 backgroundswere used for allele discrimination in MEF and M-iPS cells.

Primers: Bis-Oct4: (SEQ ID NO: 25) Fw: TTAGAGGATGGTTGAGTGGGTTTGTAAGGAT (SEQ ID NO: 26) Rev: CCA ATCCCACCC TCTAACCTTAACCTCTAA (these primers amplify only the endogenous  copy of Oct-4.) Bis-Nanog(SEQ ID NO: 27) Fw: TAAATTGGGTATGGTGGTAGATAAGTTTGG  (SEQ ID NO: 28)Rev: TAAAAAACATCCTCTAATCTAAAAACATCC  Bis-Snrpn (SEQ ID NO: 29)Fw: ATTGGTGAGTTAATTTTTTGGA  (SEQ ID NO: 30) Rev: ACAAAACTCCTACATCCTAAAA 

Generation of Chimeras

Chimeras were produced by injecting (B6-JF1) M-iPS cells into CD1blastocysts that were subsequently implanted into pseudo-pregnant CD1females. M phase extract-treated iPS clones were sexed by karyotyping.

Purification and Analysis of Chromatin Fractions

Permeabilized MEF nuclei were incubated in M phase Xenopus egg extractsfor 40 min, diluted in 5 volumes of XB buffer and pelleted bycentrifugation at 500 g through a 0.7M sucrose cushion for 10 min.Nuclear pellets were resuspended in XB with 0.2% Triton X-100 andincubated on ice for 5 min. Chromatin pellets were recovered bycentrifugation at 5000 g for 5 min, adjusted in Laemmli buffer andanalyzed by SDS-PAGE. Western blot analysis was performed using thefollowing antibodies: anti-ser10 phosphorylated histone H3 (Ozyme,9701S), anti-histone H3 (Abcam, ab1791), anti-HP1α (Millipore, MAB3584or 2616), anti-histone variant H3.3 (Abcam, ab62642), anti-Lamin B1(Abcam, ab16048), anti-H3K4me2 (Abcam, Ab7766), anti-H3K4me3 (Abcam,Ab1012), anti-H3K9me2 (Millipore, 07-441), anti-H3K9me3 (Upstate),anti-H4K20me3 (Abcam, ab9053), anti-H4K8acetyl (Abcam, ab1760),anti-H3K27me3 (Millipore, 07-449), anti-H3K9acetyl (Abcam, ab4441) andanti-acetyl H3 (Millipore 06-599).

Viral Integration

All the cell populations (not infected MEFs, infected MEFs and MEFs thathave been infected, permeabilized and incubated with M phase Xenopus eggextracts or buffer) were harvested 21 days after infection and total DNAwas extracted with the DNEasy kit according to the manufacturer'sprocedure. Quantitative PCR was then performed as described above.Quantification data were normalized to the average of two genomicregions and relative to the DNA of not infected MEFs.

Primers: DNA Oct4 (SEQ ID NO: 31) Fw: aagttggcgtggagactttg (SEQ ID NO: 32) Rev: tctgagttgctttccactcg  DNA Klf4 (SEQ ID NO: 33)Fw: gctcctctacagccgagaatc  (SEQ ID NO: 34) Rev: atgtccgccaggttgaag DNA Sox2 (SEQ ID NO: 35) Fw: tcaagaggcccatgaacg  (SEQ ID NO: 36)Rev: ttgctgatctccgagttgtg  DNA cMyc (SEQ ID NO: 37)Fw: gctggagatgatgaccgagt  (SEQ ID NO: 38) Rev: atcgcagatgaagctctggt DNA genomic1 (SEQ ID NO: 39) Fw: gtcaccgtttgtgccgaa  (SEQ ID NO: 40)Rev: agctgaaatgagaccgattatgg  DNA genomic2 (SEQ ID NO: 41)Fw: gagtcaaagagtggtgaaggagttagt  (SEQ ID NO: 42)Rev: agctgacgggccttctaagtc 

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The invention claimed is:
 1. A method for obtaining fully reprogrammedmammalian iPS cells comprising: incubating at least one permeabilizednucleus of a mammalian induced pluripotent stem (iPS) cell, or at leastone permeabilized, isolated mammalian iPS cell comprising said nucleus,with an isolated extract of Xenopus oocytes, said oocytes blocked atmetaphase II of meiosis, wherein said extract comprises EGTA; whereinthe isolated mammalian iPS cell is produced by reprogramming of amammalian somatic cell with retroviral vectors encoding Oct-4, Sox-2,Klf-4 and c-myc; wherein the method produces a higher yield of fullyreprogrammed iPS cells as compared to mammalian iPS cells produced bythe same method but without incubation in said extract.
 2. A compositioncomprising: at least one permeabilized nucleus of a mammalian inducedpluripotent stem (iPS) cell, or at least one permeabilized, isolatedmammalian iPS cell comprising said nucleus: wherein the isolatedmammalian iPS cell is produced by reprogramming of a mammalian somaticcell with retroviral vectors encoding Oct-4, Sox-2, Klf-4 and c-myc; anisolated extract of Xenopus oocytes, said oocytes blocked at metaphaseII of meiosis, wherein said extract comprises EGTA.